One or more embodiments of the present invention relates to a super-resolution microscopy method and optical device, i.e. with a resolution below the diffraction limit, for three-dimensional localization of one or more particles.
Mankind's understanding of the dynamic architecture of cells is in the process of being completely transformed by technological developments allowing individual molecules to be detected optically in living systems. By virtue of ultrasensitive measuring methods, it is now possible to count, localize and follow the movement of biological molecules in their cellular environment (see for example B. Huang et al. “Super-resolution fluorescence microscopy” Annu Rev Biochem 78, 993 (2009)). In this way, it is possible to analyze the composition, structure and spatial dynamics of molecular complexes with a spatial resolution of a few nanometers and a temporal resolution reaching a millisecond. This opens a window onto a complex molecular organization that could not previously be studied with the microscopy techniques conventionally used in biology and biochemistry. Remarkably, techniques for imaging single molecules have already found applications in fields beyond fundamental research, especially in the important field of DNA sequencing (T. D. Harris et al. “Single-molecule DNA sequencing of a viral genome”, Science 320, 106 (2008)). In the medium term, it seems likely that their use will spread to the fields of diagnostics or molecular targeting, fields in which an ultrasensitive detection capacity is a major advantage. There are therefore major scientific and industrial incentives to develop effective approaches to imaging at the scale of individual molecules.
Generally, our cells may be considered to be reactors in which a multitude of biochemical reactions take place between a no less considerable number of reactants (which, for the most part, are proteins). Within a cell, proteins assemble into reactive units that are called macromolecular complexes. The average size of protein assemblies with cellular functions typically ranges from a few nanometers for small complexes to about 100 nanometers for the largest structures such as nuclear pores. Most molecular complexes (nucleosome, RNA polymerase, ribosomes) are between 10 and 30 nm in size. The various interactions between these complexes, and the molecular modifications that result therefrom, form the network of interactions and reactivity that is the physical and chemical medium of all cellular regulation. Analysis of these networks is at the heart of our understanding of cellular processes. Most cellular dysfunctions that result in pathologies are in fact caused by a defect in the interaction or presence of one of the partners of a cellular macromolecular complex. To understand these pathologies, with the aim of effectively combating them, it is indispensable to develop measurement tools capable of providing quantitative information on the stoichiometry (molecular count) and position of protein complexes relative to one another.
At the present time, it is possible to functionalize practically any protein in an organism by adding a genetically encoded tag to it, this tag either being directly fluorescent or being able to react with a soluble fluorescent compound (see for example B. N. Giepmans et al. “The fluorescent toolbox for assessing protein location and function”, Science 312, 217 (2006)). These probes are capable of emitting a number of photons comprised between a few hundred thousand and a few million before photobleaching. In other words, tagging with a probe is equivalent to allocating a “photon budget” to a particular protein, which budget may then be used to transfer molecular information to our macroscopic world (for example by way of an amplified CCD camera). Thus, the photons emitted by a fluorescent protein especially allow its position in its cellular environment to be pinpointed with a resolution of a few tens of nanometers, or it to be followed in time, thus allowing its mobility and its interactions with the cellular system to be measured.
The ability to localize molecules is at the heart of pointillist super-resolution microscopies (known by the acronyms PALM for “photo-activated localization microscopy” or STORM for “stochastic optical reconstruction microscopy”). These microscopies, which combine nanoscale localization and control, by photoactivation, of the number of simultaneously active emitters, allow two-dimensional images of cellular samples to be obtained with a resolution of 10-50 nm, far below the conventional diffraction limit (˜250 nm in modern epifluorescence microscopes) (B. Huang et al. “Super-resolution fluorescence microscopy” Annu Rev Biochem 78, 993 (2009)). Since the resolution obtained is of the same order of magnitude as the size of the macromolecular assemblies involved, the study of relationships between the spatial organization and the function and structure of the macromolecules becomes possible.
By placing a cylindrical lens on the optical path of the signal, and by calibrating the ellipticity induced in the point spread function (PSF) of the microscope for the individual molecule, 3D STORM microscopy with an axial resolution of about 100 nm, about 2.5 times that in the perpendicular plane, was demonstrated in the laboratory of X. Zhuang at Harvard in 2008 (see for example B. Huang et al. “Three dimensional super-resolution imaging by stochastic optical reconstruction microscopy” Science 319, 810 (2008) or the patent application US 2011/0002530 in the name of the same inventors). With this technique, the axial resolution is related to the break in symmetry of the optical signal along the x- and y-axes as a function of the distance of the fluorophore from the focal plane.
A second method consists in simultaneously imaging the signal from an individual molecule in two axially separate planes. With this simultaneous “biplane” detection, the z-position of a molecule between the two planes may be determined with a precision similar to that achieved with the astigmatic approach (see for example M. F. Juette et al. “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples” Nat Methods 5, 527 (2008)); the advantage of this technique is that lateral and axial resolution are not coupled.
However, a limitation that is common to both the cylindrical-lens and biplane techniques is related to residual optical aberrations that deform the PSF and deteriorate the algorithms for laterally and axially localizing the emitting particle. Moreover, the depth (range) over which it is possible to determine the axial position of the molecule is limited (to about 1-2 μm) and is entirely fixed by the opto-mechanical elements of the apparatus, meaning that it is impossible to make rapid adjustments. This is clearly an obstacle to optimum use of the “photon budget”, this optimum use depending on the different types of fluorophore used and the biological applications in question.
Lastly, it will be noted that other less commonplace methods have been demonstrated, such as the “double-helix PSF” method (S. R. Pavani et al. “Three dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function” Proc. Natl Acad Sci USA 106, 2995 (2009)) or the “iPALM” method (G. Shtengel et al. “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure” Proc. Natl Acad Sci USA 106, 3125 (2009)). Moreover, at the present time the latter technique enables the best localization in z, but at the price of considerable experimental complexity (4pi measuring system and triple interferometric detection of emitted photons) which confines it to marginal use in biological laboratories. In addition, it remains limited to fixed samples.
One or more embodiments of the invention provides a method and a device for three-dimensional localization of emitting particles, or “emitters”, with a resolution below the diffraction limit, and in which excellent control of the point spread function of the microscope is obtained, especially allowing measurement reliability to be increased and the use of the available “photon budget” for a given emitter to be optimized.